Spermatogenic cell-specific type 1 hexokinase (HK1S) is essential for capacitation-associated increase in tyrosine phosphorylation and male fertility in mice

Hexokinase (HK) catalyzes the first irreversible rate-limiting step in glycolysis that converts glucose to glucose-6-phosphate. HK1 is ubiquitously expressed in the brain, erythrocytes, and other tissues where glycolysis serves as the major source of ATP production. Spermatogenic cell-specific type 1 hexokinase (HK1S) is expressed in sperm but its physiological role in male mice is still unknown. In this study, we generate Hk1s knockout mice using the CRISPR/Cas9 system to study the gene function in vivo. Hk1s mRNA is exclusively expressed in testes starting from postnatal day 18 and continuing to adulthood. HK1S protein is specifically localized in the outer surface of the sperm fibrous sheath (FS). Depletion of Hk1s leads to infertility in male mice and reduces sperm glycolytic pathway activity, yet they have normal motile parameters and ATP levels. In addition, by using in vitro fertilization (IVF), Hk1s deficient sperms are unable to fertilize cumulus-intact or cumulus-free oocytes, but can normally fertilize zona pellucida-free oocytes. Moreover, Hk1s deficiency impairs sperm migration into the oviduct, reduces acrosome reaction, and prevents capacitation-associated increases in tyrosine phosphorylation, which are probable causes of infertility. Taken together, our results reveal that HK1S plays a critical role in sperm function and male fertility in mice.


Introduction
Mammalian sperm leaving the male reproductive tract after ejaculation are functionally immature, exhibit progressive motility, and cannot successfully fertilize oocytes.To acquire fertilizing ability, sperm must undergo several biochemical and functional changes in the female reproductive tract, collectively known as capacitation [1,2].During capacitation, sperm switch from progressive to hyperactivated motility, and undergo the acrosome reaction [2,3].These processes allow sperm to penetrate the cumulus cell layer and zona pellucida (ZP) of the oocyte to complete fertilization [3], whereas defects in these events can finally lead to subfertility or infertility.
Hexokinase (HK) is an enzyme that catalyzes the first committed step of glycolysis, which utilizes ATP to phosphorylate glucose and produce glucose-6-phosphate (G6P).G6P can enter the glycolytic pathway for energy production, the pentose phosphate pathway (PPP) for anabolic intermediates, or be converted to glucose-1-phosphate for glycogen synthesis [4,5].In mammals, five HK isozymes (HK1, HK2, HK3, glucokinase [GCK], and HK domain-containing 1 [HKDC1]) have been identified, each with distinct tissue expression, subcellular localization and enzyme kinetics [4,6].HK1 is a ubiquitous enzyme that is present in most cell types, especially at the highest levels in brain and erythrocytes [7,8].It contains an N-terminal 20-amino acid hydrophobic sequence termed porin-binding domain (PBD) that binds HK1 to voltage-dependent anion channels (VDACs) in the outer mitochondrial membrane [4,9].However, three variant transcripts of Hk1 (Hk1_v1, Hk1_v2, and Hk1_v3) are expressed specifically in spermatogenic cells, and named spermatogenic cell-specific type 1 hexokinase (HK1S).HK1S encodes a different N-terminal 24-amino acid sequence, called spermatogenic cell-specific region (SSR), which replaces the PBD of the HK1 isoform present in somatic cells [8,10,11].HK1S is tethered in the principal piece region by a spermatogenic cell-specific muscle-type phosphofructokinase variant isozyme (PFKMS), which in turn is tightly bound to glutathione S-transferase mu class 5 (GSTM5) in the fibrous sheath (FS) [12].
Glycolysis and oxidative phosphorylation (OXPHOS) are the two major metabolic pathways producing ATP which is the primary source of energy for sperm, with glycolysis occurring in the FS of the principal piece, and OXPHOS occurring in mitochondria that are exclusively localized in the midpiece of flagellum [13,14].In some mammals, such as humans and mice, ATP is mainly produced by glycolysis [15,16], and most of the glycolytic enzymes have unique structures and functions.Some are products of alternative transcript splice variants of genes expressed in other cells, such as HK1S [8,10,17], PFKMS [12], and aldolase (ALDOA_V2) [18].Others are products of spermatogenic cell-specific genes, including glyceraldehyde 3-phosphate dehydrogenase, spermatogenic (GAPDHS) [19,20], lactate dehydrogenase-C4 (LDHC) [21], phosphoglycerate kinase 2 (PGK2) [22], two aldolase A (Aldoa)-related retrogenes (ALDOART1 and ALDOART2) [18].Several glycolytic enzymes are tightly bound to the FS of mouse sperm [23].Previous in vitro studies of mouse and human sperm indicate that glycolysis produces large amounts of ATP [16,24,25] and is required for multiple steps of fertilization, including capacitation-dependent tyrosine phosphorylation [26,27], sperm motility [16,28], and penetration of the ZP [29].In vivo studies targeting disruption of glycolytic isozyme Gapdhs, Ldhc or Pgk2 respectively result in reduced levels of ATP in sperm, disruption of sperm motility, abnormal tyrosine phosphorylation and male infertility [30][31][32], confirming that glycolytic enzymes are essential for sperm function and fertilization in mice.However, the role of HK1S in male fertility in mammals is still unknown.
Here, seeking to investigate the role of the mouse Hk1s gene in male fertility, we generate an Hk1s knockout mouse line using the CRISPR/Cas9 system.The ablation of Hk1s in mice leads to male infertility and reduces glycolytic pathway activity.In addition, IVF using Hk1s deficient sperms cannot fertilize cumulus-intact or cumulus-free oocytes but is able to successfully fertilize zona pellucida-free oocytes.Furthermore, Hk1s deficiency impairs sperm migration into the oviduct, acrosome reaction, and capacitation-associated increases in tyrosine phosphorylation.Collectively, our findings demonstrate that HK1S is indispensable for male fertility.

Expression of Hk1s in adult mice and generation of Hk1s knockout mice
We initially examined the transcript levels of the HK gene family members in sperm from the adult mouse cauda epididymis using quantitative real-time PCR (qRT-PCR).We found that Hk1s was highly expressed and Hk2 was moderately expressed in sperm, while Hk1, Hk3, Gck and Hkdc1 were undetectable in sperm (Fig 1A).We then analyzed Hk1s mRNA transcript levels in various adult mouse tissues and found that Hk1s was restricted to the testis (Fig 1B).To determine the expression pattern of Hk1s in testes from mice sampled at postnatal days (P) 1, 8, 10, 12, 14, 18, 21, 24, 30, 35 and 80, we observed a rapid increase in the Hk1s mRNA level starting from P18 and continuing to P35, the period when late pachytene spermatocytes appear and develop through spermiogenesis to produce mature sperm (Fig 1C).These results suggest that HK1S may play roles in late spermatogenesis and/or fertilization.
To more fully understand the physiological function of Hk1s, we used the CRISPR/Cas9 technology to generate the Hk1s knockout mouse model with targeted deletion of the N-terminal SSR (Fig 1D).We confirmed the successful inactivation of this gene at the genome and mRNA transcription levels (Fig 1E and 1F).Western blot for HK1 antibody (recognizing HK1 and HK1S) showed that HK1(S) exhibited similar expression level between Hk1s +/− and Hk1s −/− brain, whereas HK1(S) was absent in Hk1s −/− testis and sperm (Figs 1G and S1).Our results further show that only HK1S is highly expressed in testis and mature sperm, while HK1 has no expression.

HK1S is localized on the outer surface of FS
Immunofluorescence analysis for HK1 antibody (recognizing HK1 and HK1S, red signal), peanut agglutinin (PNA, acrosome marker) and α-Tubulin (main component of spermatid flagella) showed that the red positive signals were strongly expressed in the acrosomal region of step 3-11 spermatids and flagella of step 15-16 spermatids in the Hk1s +/− mouse testis, while the red positive signals still remain in the acrosomal regions of step 3-11 spermatids in the Hk1s −/− mouse testis.This suggests that the fluorescence signals in round spermatids may be false positive, and indicates that the HK1S protein is localized to the flagella region of step 15-16 spermatids (Figs 2A and S2), as has been found in previous studies [8,17].To confirm this, we also isolated round spermatids and elongating/elongated spermatids from adult testicle samples using STA-PUT velocity sedimentation via bovine serum albumin (BSA) density gradient (S3A Fig) and test HK1(S) expression by western blot.We found that no HK1 or HK1S is expressed in the round spermatids (S3B Fig).Therefore, the above results showed that HK1S was only localized in the flagella region of step 15-16 spermatids in mouse testis.Immunofluorescence staining revealed that HK1S protein was localized in the principal piece of sperm flagellum, which was easily distinguishable by staining the mitochondrial sheath with a MitoTracker dye in Hk1s +/− sperm.As expected, no HK1S positive signaling was found in Hk1s −/− sperm (Fig 2B).To further analyze the subcellular localization of HK1S protein, we fractionated sperm proteins into three fractions: the Triton X-100 soluble fraction containing transmembrane and cytosolic proteins, the SDS-soluble fraction containing axonemal proteins, and the SDS-resistant fraction containing proteins localized in the accessory structures such as FS and outer dense fibers [33,34].Western blot analysis indicated that HK1S was mainly present in the 1% Triton X-100 fraction in Hk1s +/− sperm (Fig 2C ), which was consistent with previous study [23].Electron microscopic immunocytochemical staining further revealed that gold particles were localized on the outer surface of FS of Hk1s +/− sperm, while Hk1s −/− sperm section had no visible gold particles (Fig 2D).These data indicated that HK1S is localized on the outer surface of FS, and it may play important roles in mature sperm.

Hk1s −/− male mice are infertile but have normal sperm morphology
To examine whether HK1S is required for fertility, individual Hk1s +/+ , Hk1s +/− , and Hk1s −/− male mice were caged with wild-type females over a period of two months.The fertility of Hk1s +/− male mice was comparable to that of Hk1s +/+ controls, but Hk1s −/− adult male mice were infertile.They mated with wild-type female mice (which formed normal vaginal plugs), but did not produce any offspring (Fig 3A).Conversely, Hk1s −/− females were fertile when they were mated with Hk1s +/+ or Hk1s +/− male mice.These results demonstrate that HK1S is essential for male fertility.
We next investigated potential mechanisms related to the infertility of Hk1s −/− male mice, by carefully monitoring the development of Hk1s +/+ , Hk1s +/− , and Hk1s −/− male mice.On reaching sexual maturity, Hk1s +/+ , Hk1s +/− and Hk1s −/− littermates had similar testis to body weight ratio, sperm densities in cauda epididymides, and gross morphology (Fig 3B -3D).Moreover, there were no obvious discrepancies in the histology and germ cell apoptosis of Hk1s +/− and Hk1s −/− testes or cauda epididymides (Figs 3E-3F and S4).A detailed investigation based on transmission electron microscopy (TEM) showed no obvious defects in the ultrastructures of Hk1s +/− versus Hk1s −/− sperm, as both genotypes exhibited normal ultrastructural features of the midpiece and principal piece (proximal and distal regions) (Fig 3G and 3H).Collectively, these observations strongly suggest that HK1S is not required for spermatogenesis and sperm production.

Hk1s deficiency reduces sperm glycolytic metabolism but does not affect sperm motility and ATP levels
To clarify the cause of impaired male fertility of Hk1s mutant mice, sperm motility was examined by real-time videos and computer-assisted sperm analysis (CASA) system.Our results found that Hk1s −/− mice showed normal sperm motility parameters in the non-capacitated conditions (Fig 4A, S1 and S2 Movies) and capacitated conditions (Fig 4B, S3 and S4 Movies).HK1S catalyzes the first step in committed step of glycolysis, therefore, we employed targeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics analysis to explore the two major energy metabolic pathways of sperm-glycolysis and tricarboxylic acid cycle (TAC) in the capacitated Hk1s +/− and Hk1s −/− sperm.Compared to Hk1s +/− sperm, glucose and pyruvate were no significant difference in Hk1s −/− sperm, due to the presence of these components in the medium (Fig 4C).However, the other intermediate metabolites involved in

Disruption of Hk1s leads to failure of sperm-ZP penetration
In order to identify the specific stage at which sperm encounter difficulties during fertilization, we performed IVF.Approximately 90% of 2-cells were found after 24 h in the Hk1s +/− sperm with cumulus-intact oocytes, and 2-cells could develop to the blastocyst stage.However, no 2-cells were found in the Hk1s −/− sperm, and fertilization rate was zero (Fig 5A and 5B).In addition, both Hk1s +/− and Hk1s −/− sperm could penetrate cumulus cell layers with cumulusintact oocytes (S6A Fig) , suggesting that Hk1s −/− sperm could penetrate the cumulus cell layers, but not fertilize cumulus-intact oocytes.Removing cumulus (cumulus-free) oocytes could not rescue impaired fertilization rates (Fig 5C and 5D).However, the fertilization rate of Hk1s −/− sperm with ZP-free oocytes was comparable to that of the control, and 2-cells could develop to the blastocyst stage at a similar ratio between Hk1s +/− and Hk1s −/− groups (Fig 5E and 5F).These findings indicate that knockout of Hk1s impairs the ability of sperm to bind or penetrate the ZP but not sperm-oocyte fusion.We thus examined the ZP-binding ability of sperm using cumulus-free oocytes and found that Hk1s −/− sperm showed normal ZP-binding ability compared with wild-type sperm (S6B Fig) .These results suggest that Hk1s −/− sperm are defective in the ZP penetration, and lead to severe fertility defects in vivo.

Disruption of Hk1s affects the sperm migration into oviduct and reduces acrosome reaction
Ejaculated sperm need to migrate from the uterus to oviducts and reach the oocytes to complete fertilization.To determine whether infertility was caused by impaired migration of the Hk1s −/− sperm in the oviduct, we analyzed sperm that arrived at oviducts of wild-type females plugged by Hk1s +/− or Hk1s −/− male mice.Histological analysis showed that Hk1s −/− sperms were much less in the colliculus of the utero-tubal junction (UTJ) 2 h after copulation, while there were numerous sperm in the nearby uterine lumen (Fig 6A).The count of all sperm in the oviduct showed that the number of Hk1s −/− sperm was less than that of control (control: 1767.2 vs Hk1s −/− : 551.0) (Fig 6B).These results suggest that disruption of Hk1s affects the sperm migration from the uterus to the oviduct.
It is widely accepted that sperm must undergo acrosome reaction to penetrate the ZP of oocyte, therefore, we analyzed the percentage of acrosome-reacted sperm in the capacitating medium from Hk1s −/− sperm.We found that the percentage of acrosome-reacted sperm did not have a significant difference between the Hk1s +/− and Hk1s −/− sperm after incubation for 10 min and 30 min.After 60 min of incubation, the percentage was significantly lower than in Hk1s +/− sperm (control: 32.2% vs Hk1s −/− : 13.0%), even after A23187 treatment, it could not reach the same level as the control group (control: 71.2% vs Hk1s −/− : 36.4%)(Fig 6C and 6D).The results were consistent with the progesterone-induced sperm acrosome reaction (control: 45.7% vs Hk1s −/− : 26.0%) (Fig 6E).These data indicate that HK1S is required for the acrosome reaction.

Hk1s deficiency disrupts tyrosine phosphorylation associated with sperm capacitation
Capacitation is the biochemical process that sperm undergo after ejaculation into the female reproductive tract and is thought to be a prerequisite for the acrosome reaction [35,36].An increase in the level of protein tyrosine phosphorylation is an important aspect of sperm capacitation [27,37], therefore we monitored the level of tyrosine phosphorylation by western blot.In the non-capacitated condition, the level of tyrosine phosphorylation was comparable between Hk1s +/− and Hk1s −/− sperm.However, in the capacitated condition, we noted that the level of protein tyrosine phosphorylation in Hk1s −/− sperm was significantly decreased compared with Hk1s +/− sperm (Fig 7A and 7B), suggesting that defects in protein tyrosine phosphorylation may contribute to the failure of acrosome reaction.In addition, HK1S was a tyrosine phosphorylated form of hexokinase [38,39], which was further confirmed by our results in the Hk1s +/− and Hk1s −/− sperm (Fig 7A and 7C, arrow).
Cyclic AMP analogs that activate protein kinase A (PKA) stimulate the same pattern of tyrosine phosphorylation at an accelerated rate [40,41].To determine if the PKA signal transduction pathway in sperm from Hk1s −/− mice is responsive to capacitation cues, we monitored protein tyrosine phosphorylation in the presence of a cAMP analog dibutyryl-cAMP (dbcAMP) and a phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) in the capacitated condition.In agreement with previous reports [41], protein tyrosine phosphorylation was exacerbated by exposure of sperm to dbcAMP and IBMX in Hk1s +/− sperm (Fig 7C).In addition, a significant increase in tyrosine phosphorylation was also observed when sperm were exposed to dbcAMP and IBMX in Hk1s −/− sperm, and it could fully recover to the same level as the control group (Fig 7D ).Taken together, these results suggest that HK1S can trigger protein tyrosine phosphorylation by mediating cAMP-PKA pathway.

Discussion
HK1S, one of the spermatogenic cell-specific variant isozymes that was reported to be specifically present in the mature sperm, has been localized to the principal piece in mice and has a novel spermatogenic cell-specific region at the N-terminus [8].Similarly, our result showed that HK1S was restricted to the flagella region of step 15-16 spermatids in mouse testis, and localized in the principal piece of sperm flagellum.In addition, HK1S in sperm was mainly solubilized with 1% Triton X-100 fraction, consistent with a previous report [23].Moreover, our more detailed observations found that HK1S was localized on the outer surface of the FS; unlike GAPDHS, ENO4, and other sperm-specific aldolase isozymes that are tightly bound to the FS [18,20,42].In the present study, we have shown that HK1S is essential for male fertility.The histological results in the testis and cauda epididymis of Hk1s −/− mice indicated that the spermatogenesis process is normal.In addition, various parameters of mature sperm in epididymis including the ultrastructures from the TEM assay all showed no difference between Hk1s −/− and Hk1s +/− male mice, suggesting that this isozyme is not required for spermatogenesis and sperm morphogenesis.Among enzymes involved in the glycolytic pathway, previous studies have shown that GAPDHS [30], PGK2 [32], and LDHC [31] are essential for male fertility and knockout of them respectively does not disrupt sperm structure, which is similar to the phenotypes of knocking out Hk1s.
Glycolysis is the main source of ATP in sperm of mice and, presumably, of many other mammalian species to provide the energy required for the flagellar activity that produces sperm motility [16].The targeted disruption of Gapdhs [30], Pgk2 [32], or Ldhc [31] in male mice severely reduced sperm motility and ATP levels.Our results show that the detailed intermediates involved in glycolysis are significantly downregulated in Hk1s −/− sperm.In contrast, Hk1s −/− sperm have normal motility and ATP levels.Amara et al. found other metabolic pathways such as fatty acid β-oxidation (FAO) by proteomic studies of human sperm may also contribute to the production of energy [43].Moreover, treating the stallion sperm with carnitine palmitoyl transferase 1, a rate-limiting enzyme of β-oxidation, can lead to decreased sperm motility [44].Knockout of Slc22a14 disrupts sperm FAO activity and leads to male infertility in mice [14].Collectively, the production of ATP in the male gamete may be achieved via pathways other than glycolysis and OXPHOS in Hk1s −/− sperm.
To further seek the cause inducing the infertility of Hk1s −/− male mice, IVF was performed.We found that Hk1s −/− sperm were unable to fertilize cumulus-intact or cumulus-free oocytes, but they were able to fertilize ZP-free oocytes at a similar ratio comparable to Hk1s +/− sperm.On the other hand, Gapdhs −/− , Pgk2 −/− , or Ldhc −/− sperm showed low fertilization rates (<10%) when used in IVF assays with cumulus-oocyte complexes [31,41].However, when the ZP was removed or drilled, the number of these oocytes fertilized increased by one-third observed with controls, suggesting that it implies a defect in sperm-egg fusion by Gapdhs −/− , Pgk2 −/− or Ldhc −/− sperm.These discrepancies may be related to the normal sperm motility and ATP levels in the Hk1s −/− sperm.In addition, Hk1s −/− sperm did not undergo the protein tyrosine phosphorylation changes characteristic of capacitation, which was similar to Gapdhs −/ − , Pgk2 −/− or Ldhc −/− sperm [31,41].Cyclic AMP analogs that activate PKA stimulate the same pattern of tyrosine phosphorylation at an accelerated rate [41].The level of the protein tyrosine phosphorylation had a significant increase in Hk1s −/− sperm exposed to dbcAMP and IBMX, which promotes tyrosine phosphorylation under normal conditions by stimulating PKA and inhibiting phospho-diesterases.In contrast, Gapdhs −/− or Pgk2 −/− sperm did not show an increase the tyrosine phosphorylation in capacitating medium with dbcAMP and IBMX [41].These results suggest that the pathway mediating PKA triggering of tyrosine phosphorylation is responsive in Hk1s −/− sperm, but is unresponsive in Gapdhs −/− or Pgk2 −/− sperm.
According to a previous study, the release of PKA from A-Kinase anchoring protein (AKAP) in capacitated sperm promotes a sudden Ca 2+ influx that starts in the principal piece, and propagates to the head triggering the acrosome reaction [45].In our study, we found that HK1S is localized on the outer surface of FS and not expressed in the acrosome, but the percentage of Hk1s −/− acrosome-reacted sperm had a significantly lower not only after 60 min of incubation in the capacitating medium but also after physiological agonists treatment, such as calcium ionophore A23187 or progesterone, compared to the control group.These results suggest that defects in the cAMP-PKA pathway may impact the AR and contribute to the failure to fertilize ZP-intact oocytes.On the other hand, Gapdhs −/− or Pgk2 −/− sperm did not show defects in ionophore-induced AR, however, their sperm motility and ATP levels were markedly reduced.This indicates defects in energy metabolism may contribute to the failure of Gapdhs −/− or Pgk2 −/− sperm to fertilize ZP-intact oocytes.
In summary, our study demonstrated that HK1S plays a crucial role in male fertility in mice.Its deletion leads to defects in the number of sperm entering the oviduct, acrosome reaction, cAMP-PKA pathway, and protein tyrosine phosphorylation.Although the levels of sperm motility and ATP were relatively normal in Hk1s -/-sperm, the detailed intermediates involved in glycolysis were significantly downregulated, indicating that the HK1S is essential for mouse sperm glycolysis.Furthermore, three unique human type 1 hexokinase mRNAs exhibit testis-enriched expression [46], and these functions may be conserved in humans as well.By identifying HK1S-associated molecules and their functions, we may gain a better understanding of the causes of human male infertility.

Ethics statement
All animal experiments were approved by the Chinese Ministry of Health national guidelines, and performed in accordance with institutional regulations of the Institutional Animal Care and Use Committee at the National Institute of Biological Sciences, Beijing (#SYXK [jing] 2023-0007).

Animals
All mice in this study were C57BL/6 strains.Mice were housed in the National Institute of Biological Sciences, Beijing.The gene-modified mice generated in this study were available through Transgenic Animal Center, National Institute of Biological Sciences, Beijing.

Generation of Hk1s knockout mice
Hk1s −/− mice were generated using the CRISPR/Cas9 technology.The sgRNAs were prepared using MEGAshortscript T7 Transcription kit (AM1354, Ambion, USA) according to the manufacturer's instructions.DNA fragments containing the coding exon 3-4 of the Hk1s (N-terminal SSR) and two homology arms were used as donor templates.After co-incubation of Cas9 protein and sgRNAs, the Cas9-sgRNA complex and donor templates were injected into C57BL/6 zygotes.Injected zygotes were transferred into pseudopregnant CD1 female mice (20-30 zygotes per pseudopregnant mice).The targeting strategy, including the sgRNA sequences and the knockout alleles obtained, are depicted in Fig 1D .The primers used for genotyping were listed in the S1 Table.

Fertility assessment
Adult male mice (Hk1s +/+ , Hk1s +/− , Hk1s −/− ) were caged for 2 months with wild-type females.Copulation was confirmed by checking for vaginal plugs.The number of litters and the average number of progeny per litter were analyzed.

Isolation of spermatids using STA-PUT velocity sedimentation
Round spermatids and elongating/elongated spermatids were isolated from adult Hk1s +/− or Hk1s −/− testes using STA-PUT velocity sedimentation according to previous study [48].Briefly, the seminiferous tubules were digested in 10 ml Krebs (KH 2 PO 4 2 mM, NaCl 119.7 mM, MgSO 4 �7H 2 O 1.2 mM, dextrose 13.9 mM, CaCl 2 �2H 2 O 1.3 mM, KCl 4.8 mM, NaHCO 3 25.2 mM) with 1 mg/ml collagenase IV (40510ES60, Yeasen, China) and 0.2 mg/ml DNase I (10608ES80, Yeasen, China) at 34˚C for 20 min.Then tubules were washed with 5 ml Krebs twice and were subsequently digested with 10 ml 0.05% trypsin (25300-062, Gibco, USA) containing 0.3 mg/ml DNase I at 34˚C for 8 min to prepare single-cell suspensions.Enzymatic digestion was quenched with 1 ml of 10% fetal bovine serum (FBS) (164210, Procell, China).Cell suspensions were sieved through a 40 μm cell strainer (352340, BD Falcon, USA).The cell pellet was collected by centrifugation at 2500 rpm for 5 min at 4˚C and resuspended in 3 ml Krebs containing 0.5% BSA.To prepare 1-3% linear BSA gradient, add 5 ml of 1% BSA solution to the bottom of the tube and gradually slowly add 5 ml of 2% and 3% BSA solution to the bottom of the tube.Then, cell suspension was gently loaded onto a 1-3% linear BSA gradient and separated by sedimentation velocity at unit gravity for 1.5 h on ice.Enriched germ cell fractions were carefully collected per tube, starting from the top of the BSA gradient.Number the tubes in the same order as the fractions collected (No. [1][2][3][4][5][6][7][8].No.1-3 and No.5-8 cell fractions were respectively pooled together.The purities of these two cell samples were evaluated and identified by their morphological characterization and immunofluorescence analysis with DAPI and PNA.

Western blot
Testes and sperm were lysed in 1% Triton X-100 lysis buffer (50 mM NaCl, 20 mM Tris�HCl, pH 7.5) with 1: 10 protease inhibitor cocktail (#04693116001, Roche, Switzerland).Homogenized lysates were rotated for 60 min at 4˚C, and centrifuged at 13000 rpm for 20 min at 4˚C.The protein concentrate of supernatants was determined using bicinchoninic acid (BCA) protein assay (#23225, Thermo Fisher Scientific, USA).The equal quality of proteins of each sample was electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (IPVH00010, MilliporeSigma, USA).The membrane was blocked with 5% skim milk in TBST (Tris-buffered saline containing 0.1% Tween-20) for 2 h at room temperature, and respectively incubated with primary antibodies diluted in antibody diluent (WB500D, NCM Biotech, China) at 4˚C overnight.The PVDF membrane was then washed three times in 0.1% Tween-20 in TBST and incubated with secondary antibodies for 60 min at room temperature.After washing with TBST, the membrane was treated with the Pierce ECL 2 western blot Substrate (#34577, Thermo Fisher Scientific, USA), and signals were detected by XBT X-ray film.
For TUNEL analysis, assays were carried out using the In Situ Cell Death Detection Kit (#11684795910, Roche, Switzerland) following the manufacturer's instructions.Images were acquired using confocal microscope Zeiss LSM800 or Nikon SIM A1 and microscope Olympus VS120.
Transmission electron microscopy (TEM) TEM was performed at the Centre for Electron Microscopy of the National Institute of Biological Sciences, Beijing, following standard protocols.Briefly, the sperm were fixed with 2.5% glutaraldehyde (G5882, Sigma-Aldrich, USA) at 4˚C for overnight, followed by secondary fixing with 1% OsO 4 for 60 min on ice.Samples were then dehydrated through a series of ascending acetone solutions by the progressive lowering temperature method.The samples were then infiltrated and embedded in SPI-Pon 812 resin (Electron Microscopy Sciences).90 nm ultrathin sections were prepared using an ultramicrotome (Leica EM UC7, Leica Microsystems, Germany), and stained with 3% uranyl acetate in 70% methanol/H 2 O for 7 min, followed by Sato's lead for 2 min.Images were obtained on a TECNAI spirit G2 (FEI) transmission electron microscope at 120 kV.

Immuno-electron microscopy (IEM)
Sperm were centrifuged and frozen by high-pressure freezing using a COMPACT 01 apparatus (M.Wohlwend GmbH, Switzerland) and then dehydrated by a freeze-substitution fixation process (AFS2, Leica, Germany) with 0.1% uranyl acetate in acetone.Subsequently, sperm were embedded in LR white resin, and 90 nm sections on nickel mesh were used for immunolabeling.The sections were washed in PBS for 5 min and then blocked in 2% BSA for 10 min at room temperature, and incubated with rabbit anti-HK1 (1: 20, #2024, Cell Signaling Technology, USA) in 2% BSA at 4˚C overnight.Samples were washed three times with PBST and incubated with goat anti-rabbit IgG conjugated to 10 nm gold particles (1: 100, G7402, Sigma-Aldrich, USA) for 60 min at room temperature.After PBS wash, the sections were post-stained with 3% uranyl acetate in 70% methanol/H 2 O for 7 min, followed by Sato's lead for 2 min.Images were obtained on a TECNAI spirit G2 (FEI) transmission electron microscope.

Analysis of acrosome reaction rates
Mouse cauda epididymis was pierced and incubated in 400 μl TYH medium for 10 min, 30 min, and 60 min at 37˚C under 5% CO 2 .Using two ways to induce the acrosome reaction: 1) Sperm were released from the cauda epididymis in TYH medium for 10 min and then treated with a calcium ionophore A23187 (C7522, Sigma-Aldrich, USA) at a final concentration of 10 μM for 50 min.2) Sperm were capacitated in TYH medium at 37˚C under 5% CO 2 for 30 min.Then capacitated sperm were incubated with 5 μM progesterone (P0130, Sigma-Aldrich, USA) in TYH medium at 37˚C for 30 min.The sperm were fixed in 2% PFA, spotted onto glass slides and air-dried.Acrosome status was evaluated by staining with Coomassie Brilliant Blue (0.22% R-250, 50% methanol, 10% glacial acetic acid, 40% water) for 5 min.For each glass slide, at least three different fields and 300 sperm were examined.

RNA purification and quantitative real-time PCR (qRT-PCR)
Tissues or sperm were homogenized in TRIZOL reagent (#15596026, Invitrogen, USA) and chloroform was added to extract twice.After centrifuging, the upper aqueous phase was transferred into isopropanol followed by another centrifuging to obtain RNA pellet.Pellet was washed two times using 75% ethanol, air-dried, and solubilized in nuclease-free water.Reverse transcription was performed using PrimeScript RT reagent kit with gDNA Eraser (RR047A, TaKaRa, China) according to the manufacturer's instructions.qRT-PCR was performed using SYBR Green master mix (DRR420A, TaKaRa, China) using Bio-Rad CFX96 Real-Time System.Relative mRNA expression levels were calculated using the comparative CT method (normalized to the Gapdh).All of the experiments were repeated independently at least three times.The primers used are listed in the S1 Table.

LC-MS-based targeted metabolomics analysis
Sperm were isolated from cauda epididymis and incubated for 2 h under capacitated condition in 500 μl TYH medium at 37˚C in 5% CO 2 .After centrifugation at 13000 rpm, 4˚C for 5 min, the pellet was washed three times with PBS, added 1 ml precooled lysis buffer (methanol: acetonitrile: water = 40: 40: 20[vol/vol]), disintegrated by physical shock for 3 min and incubated at -80˚C for 60 min.After centrifugation at 13000 rpm, 4˚C for 20 min, the supernatant was concentrated in a vacuum evaporation.Sample dry powder was stocked at -80˚C until further analysis.

Sperm ATP level measurements
Sperm ATP levels were measured using CellTiter-Glo Luminescent Cell Viability Assay (G7570, Promega, USA) [49].Sperm were incubated in TYH medium at 37˚C in 5% CO 2 in humidified air and assayed after 10 min and 2 h, and adjusted to about 5x10 6 sperm/ml.50 μl of each sample were transferred to wells in a white 96-well microtiter plate.Subsequently, 50 μl CellTiter-Glo Reagent was added, and the mixture was shaken in the dark for 10 min at room temperature (24-26˚C), Luminescence was recorded with a Tecan GENios Pro plate reader.

Analysis of sperm migration into the oviduct
Superovulated wild-type female mice were caged with Hk1s +/− and Hk1s −/− males and checked for the formation of vaginal plugs every 30 min.Female mice were killed 2 hours after vaginal plug formation.Bilateral oviducts were removed and flushed out the sperm with TYH using a 26-gauge needle.The number of sperm was counted using a hematocytometer.For H&E analysis, uterus and oviduct junction of plugged females was excised and fixed 2 h after coitus.
Serial sections containing UTJ were made from paraffin-embedded tissue, stained with H&E, and examined for the presence of sperm under a bright-field microscope system (Leica Microsystems, Germany).

In vitro fertilization (IVF)
Sperm collected from the cauda epididymis were incubated in a drop of TYH medium for 60 min at 37˚C under 5% CO 2. Oocytes were collected from the superovulated females, treated with hyaluronidase (H3757, Sigma-Aldrich, USA) for 10 min to remove the cumulus cells (cumulus-free oocytes) or with Tyrode's salt solution (T1788, Sigma-Aldrich, USA) for 1 min to remove the ZP.Sperm were added to the TYH drops that contain cumulus-intact, cumulusfree, or ZP-free oocytes at a final density of 1x10 6 sperm/ml and incubated at 37˚C under 5% CO 2. Two-cells were counted the next day using Hoffman Modulation Contrast microscope (OLYMPUS IX71).And fertilization rate was calculated as the ratio between the number of pronuclear-stage embryos and the total number of pronuclear-stage embryos plus oocytes recovered [50].For sperm penetration and binding assays, sperm were added to the TYH drops that contain cumulus-intact or ZP-free oocytes at a final density of 1x10 5 sperm/ml and incubated at 37˚C under 5% CO 2. After 60 min of coincubation, oocytes were washed gently with TYH medium to remove free sperm and the sperm binding loosely.

Fig 1 .
Fig 1.Expression pattern of Hk1s in mice and generation of Hk1s −/− mice.(A) qRT-PCR analysis of transcript levels of members of the HK gene family in mouse sperm.Mice number (n = 3).(B) qRT-PCR analysis of Hk1s mRNA levels in various organs of adult mice.Mice number (n = 3).(C) qRT-PCR analysis of Hk1s mRNA levels in mouse testes of indicated ages.Mice number (n = 3).(D) Schematic diagram showing the gene structure of Hk1s and the CRISPR/Cas9 system used to generate the knockout allele.The upper panel shows the region of the Hk1s locus that was targeted.Two gRNAs were used to achieve deletion of the SSR genomic fragment (6399bp).The frameshift-mutated sequences of the knockout allele and Sanger sequencing are shown in the middle and lower panels.The locations of the gRNAs and primers (F1, R1, and R2) are indicated.(E) Genotyping of Hk1s −/− mice by primers indicated in (D); the sizes of PCR products are shown on the left.(F) qRT-PCR analysis of Hk1s mRNA levels in Hk1s +/− and Hk1s −/− sperm.Mice number (n = 3) per genotype.Error bars: SEM.Statistics, Student's t-Test.***, P < 0.001.(G) Western blot analysis of HK1 and HK1S protein levels in Hk1s +/− and Hk1s −/− brain and sperm.α-Tubulin was used as a loading control.https://doi.org/10.1371/journal.pgen.1011357.g001

Fig 6 .
Fig 6.Disruption of Hk1s affects the sperm migration into oviduct and reduces acrosome reaction.(A) H&E staining showed the migration of sperm from uterus into oviducts.Uterus, colliculus of the UTJ, and sperm ejaculated into uterus are shown in the middle panels.Left and right panel insets show higher magnification.Arrows show sperm.More than three females plugged by a male of each genotype were detected and representative images are shown.Left and right bar: 20 μm, middle bar: 200 μm.(B) Sperm number in the bilateral oviducts of plugged females were counted and analyzed.Mice number (n = 5-6) per genotype.Error bars: SEM.Statistics, Student's t-Test.***, P < 0.001.(C) Non-AR and AR sperm were stained by Coomassie Brilliant Blue R-250, and acrosome disappeared in the AR sperm.Black arrowheads indicate the disappeared acrosome; Red arrowheads indicate the intact acrosome.Scale bar: 20 μm.(D) The percentage of the AR in Hk1s +/− and Hk1s −/− sperm, which was incubated after 10 min, 30 min, 60 min in the capacitating medium.Calcium ionophore A23187 was added to induce the AR for 60 min.Mice number (n = 3) per genotype.Error bars: SEM.Statistics, Student's t-Test.**, P < 0.01; ns: non-significant.(E) The percentage of the AR was induced by progesterone (P4) in capacitated Hk1s +/− and Hk1s −/− sperm.Mice number (n = 3) per genotype.Error bars: SEM.Statistics, Student's t-Test.**, P < 0.01.https://doi.org/10.1371/journal.pgen.1011357.g006